Epigenetic marks are chemical modifications of chromatin structure, and different marks are frequently associated with different patterns of gene expression. Alterations of epigenetic profiles are a well-established feature of cancer cells. One of the most prominent alterations is aberrant DNA hypermethylation at promoters, which has been associated with inappropriate gene silencing, such as tumor suppressor inactivation, and is hypothesized to serve as an alternative to traditional DNA mutations in promoting many forms of cancer. However, whether dysregulated epigenetic changes are a primary cause that drives malignant gene expression or rather a downstream effect of other changes in the cell remains a central question in cancer epigenetics. The trans-complementation assay developed in Dr. Bruce Lahn's Lab is a powerful tool that is capable of ascertaining whether epigenetic mechanisms play a causative role in gene silencing. Previous work in the Lahn lab has shown that this assay can identify a mode of gene silencing that has since been termed "occlusion". More specifically, occlusion refers to a state of transcriptional competency whereby the silent state of a gene is enforced by chromatin modifications of the gene even when transcriptional activators of that gene are present in the cellular milieu. For an occluded gene, therefore, chromatin state acts causally in silencing expression rather than silencing resulting from other factors present in the cell. Since trans-complementation can identify the causative role of epigenetic mechanisms in gene silencing, this assay serves as an ideal platform for addressing the contribution of altered epigenetic states, especially altered DNA methylation, in cancer initiation and progression. We hypothesize that changes in gene occlusion status underlie altered expression patterns of at least some genes in tumor cells. Furthermore, we propose to determine whether aberrant DNA methylation is one of the biochemical mechanisms responsible for changed occlusion status in tumor cells. Thus, the specific aims of my research are (1) to construct an occlusion map of tumor cells and (2) to determine if aberrant DNA methylation underlies altered gene occlusion in tumor cells. Using the trans-complementation assay recently developed in the Lahn lab, we will generate maps of occluded genes in tumor cells and their normal counterpart cells. For this, we will employ a well-defined genetic model of cancer, the N-RASQ61K INK4a-/- mouse model of metastatic melanoma. More specifically, we will determine if primary melanoma cells and metastatic tumor cells display defects in gene occlusion as compared to normal melanocytes. We will also perform genome-wide mapping of the DNA methylome in the aforementioned cell types. These analyses will help to address whether aberrant DNA methylation acts as a biochemical mechanism underlying altered gene occlusion in tumor cells. PUBLIC HEALTH RELEVANCE: In recent years, increasing attention has been placed on the role that altered epigenetic states might play in cancer development, partly because unlike genetic changes, epigenetic changes are potentially reversible. However, a causal link connecting epigenetic events to altered gene expression and cancer initiation/progression is yet to be established. Therefore, identifying whether epigenetic alterations act as the source of aberrant gene expression in cancer cells would be a critical advance for the field of cancer biology, and has the potential to lead to better diagnosis and treatment strategies.